The presence of immature granulocytes (IG) in peripheral blood is potentially important information which indicates enhanced bone marrow activation. Besides the obvious significance of blasts for the diagnosis of leukaemia, the promyelocyte, myelocyte and metamyelocyte stages of myeloid maturation may indicate systemic inflammatory stress or leukaemic reactions. The determination of immature granulocytes is routinely done by visual microscopy, which requires manual review of each blood sample smear, and is a labor intensive and time consuming task.
Currently, several high end hematology analyzers which utilize optical, fluorescence and impedance measurements to provide automated determination of immature granulocytes of the blood samples. However, these instruments and their detection systems are expensive, and not suitable for low cost analyzers. Therefore, there is a need for automated and inexpensive determination of immature granulocytes and reduction of manual review rate.
On the other hand, the presence of nucleated red blood cells (NRBCs), or referred to as erythroblasts, one type of immature red blood cells, in the peripheral blood is also important information for diagnosis of certain diseases, such as anemia, and leukemia, etc. Therefore, it is of clinical importance to measure nucleated red blood cells in blood samples. Traditionally, differentiation and enumeration of NRBC are performed manually. The process involves the smearing of a blood sample on a microscope slide and staining the slide, followed by manual visual analysis of the individual slide. The NRBC concentration is reported as numbers of NRBC per 100 white blood cells. Usually, 200 white blood cells and the numbers of NRBC present in the same region on a blood smear are counted and the numbers are divided by 2 to express the NRBC concentration as the numbers of NRBC/100 WBC. This approach is time-consuming as well as being subjective to the interpretation of the individual analyzing the slide.
In recent years, several fluorescence flow cytometry methods have been developed for differentiating NRBCs. These methods utilizes specific nuclear staining technique to distinguish NRBCs from other cell types because it is difficult to differentiate NRBCs based on their electronic or optical properties.
U.S. Pat. No. 6,410,330 (to Li et al) discloses a method for differentiation of nucleated red blood cells. The method includes the steps of lysing red blood cells of a blood sample with a lytic reagent, measuring nucleated blood cells by DC impedance measurement in a non-focused flow aperture, differentiating nucleated red blood cells from other cell types, and reporting nucleated red blood cells in the blood sample.
U.S. Pat. No. 6,472,215 (to Huo et al) teaches a method of differentiating nucleated red blood cells by lysing a first aliquot and a second aliquot of a blood sample separately with a first lysing reagent system and a second lysing reagent system; measuring the first sample mixture in a flow cell by DC impedance, radio frequency, and light scatter measurements; measuring cell distributions and counting remaining blood cells in the second sample mixture by DC impedance measurements in a non-focused flow aperture; analyzing blood cell distribution patterns obtained from measuring the first sample mixture and from measuring the second sample mixture respectively; and further performing a combined analysis to differentiate NRBCs from other cell types and determine numbers of NRBCs in the blood sample.
Furthermore, a well known problem with NRBC containing samples is erroneous white blood cell count (WBC) reported by hematology analyzers on these samples. Since the nuclear volumes of NRBC are close to those of white blood cells, and they are commonly counted as white blood cells on hematology analyzers which measure the sizes of blood cells, resulting an elevation of WBC. Therefore, correction of NRBC contribution to the WBC reported from hematology analyzer is required for samples containing NRBC. Current practice in the clinical laboratory is to subtract the numbers of NRBC obtained by manual count from the WBC count reported by the hematology analyzers. This is time consuming and error prone.
Based on foregoing, there exists a need for a simple and less costly analysis method for differentiating and enumerating nucleated red blood cells. Furthermore, it is desirable to provide automated determination of immature granulocytes together with the determination of nucleated red blood cells in one concurrent test.